Project 3: Immune regulation in visceral leishmaniasis will continue to study the immunologic defects associated with human visceral leishmaniasis with a view toward developing immune based therapies that will reverse the immunosuppression associated with active disease. The studies will continue to focus on the nature and mode of action of regulatory cells and suppressive cytokines that are present in lesional tissue and peripheral blood during active disease. Four specific aims are proposed: Aim 1 will undertake comprehensive immune response profiling of peripheral, whole blood cells from active and clinically cured VL patients at the transcriptional and protein levels to identify correlates of immunity or progressive disease. Aim 2 will test the effect of immune-modulation on parasite survival ex vivo. Splenic aspirate cells obtained for diagnostic purposes will be used to reveal a direct effect of endogenous IL-10 as well as other activating and deactivating cytokines on parasite growth or killing in a target organ by taking advantage ofthe fact that the aspirate contains infected macrophages as well as the regulatory and effector cells thought to be relevant to the infection outcome. Aim 3 will explore in detail the regulation of IL-10 production by T cells in human VL, focusing on IL-27 and lL-21 signaling in the induction and amplifcation of the T cell IL-10 response. Elevated IL-10 is associated with many chronic infections in humans, including HIV, TB, and malaria, yet no studies on the regualtion of human IL-10 in an infectious disease setting have been reported. Aim 4 will investgate the role of CD8+ T cells in human VL to determine if they contribute a suppressive or pathologic function. Splenic CD8+ T cells from VL patients have been found to be enriched in IL-10 and in inhbitory signaling molecules, PD-1 and CTLA-4. No CD8 functional studies in VL have been previously described. RELEVANCE (See instructions): Project 3. The studies proposed in Project 3 may reveal immunosuppressive pathways that account for the severe progression and fatal outcome of untreated VL, and may identify novel targets of immune based therapies to enhance current treatments for VL.